Long, non-coding RNAs (lncRNAs) have emerged as important regulators of chromatin structure and function. Vertebrate lncRNAs have been closely associated with epigenetic silencing by recruiting epigenetic repressors to target genes. The specific hypothesis behind the proposed research is that the novel lncRNA Scirocco (SCIR) controls cell differentiation and proliferation in development and cancer. The specific aims are designed to provide a comprehensive assessment of the role of SCIR in cell differentiation and proliferation in development and disease. Aim 1. To dissect the interaction of SCIR with EZH2: SCIR interacts with the EZH2 subunit of PRC2 in vitro and the interaction of SCIR with EZH2 evicts PRC2 from chromatin. How SCIR interacts with EZH2 remains unclear. The RNA and protein motifs involved in the association of SCIR with EZH2 will be uncovered by in vitro RNA-protein binding assays, which investigate the interaction of EZH2 with mutant SCIR transcripts and vice versa. To assess whether the identified RNA and protein motifs support the EZH2-SCIR interaction in vivo, we shall investigate the association of EZH2 with SCIR in cells, which express mutant SCIR transcripts or mutant EZH2 proteins. Aim 2. To dissect the role of SCIR in development: Our data suggest that all-trans retinoic acid mediated activation of SCIR results in activation of HOXA1 and HOXA2 and culminates in cell differentiation and proliferation. How RA activates SCIR transcription and how the SCIR-mediated activation of HOXA1 and HOXA2 controls cell proliferation and differentiation remains unknown. We shall combine RNA- and ChIP- sequencing assays with functional assays to identify target genes for HOXA1 and HOXA2. To uncover the role of SCIR in development, we shall investigate the development SCIR-deficient cells and mice. We shall investigate whether the inactivation of components of the RA signal transduction pathway attenuates SCIR transcription. Aim 3: To dissect the role of SCIR in cancer: We have detected aberrant transcription of SCIR in primary breast and lung cancer and demonstrated that SCIR controls the proliferation, migration and invasiveness of lung and breast cancer cells. Our results suggest that anomalous SCIR transcription is involved in initiation and progression of cancer. We shall investigate whether the ectopic expression of SCIR causes cancer in mice. Accomplishing the three aims of this proposal will support he role of SCIR in development and cancer by providing evidence that the lncRNA SCIR directly interacts with epigenetic repressor EZH2 (Aim 1), elucidating the role of SCIR in cell differentiation and proliferation (Aim 2), and uncovering the role of SCIR in initiation and progression of cancer (Aim 3).